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. 2017 Dec 13;8:2116. doi: 10.1038/s41467-017-02029-7

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — OTUD1 deubiquitinates SMAD7 in vivo and sustains SMAD7 stability. a IB of total cell lysate (TCL) and immunoprecipitates derived from HEK293T cells transfected with HA-Ub, Flag-SMAD7, Myc-OTUD1-wt/CA and treated with control DMSO or MG132 (5 μM for 4 h) as indicated. b, c IB of TCL and immunoprecipitates derived from HEK293T stably expressing HA-Ub and transfected with Flag-SMAD7, Myc-RNF12, and Myc-OTUD1-wt/CA (b) or depleted for OTUD1 with shRNA (#1 and #2) c as indicated. Poly-ubiquitinated SMAD7 was immunoprecipitated with anti-Flag M2 beads and analyzed by IB with anti-HA-Ub antibody. d IB of TCL and immunoprecipitates derived from OTUD1 ^+/+ and OTUD1 ^−/− HEK293T cells stably expressing HA-Ub and restored with or without Myc-OTUD1 expression plasmid. Endogenous poly-ubiquitinated SMAD7 was immunoprecipitated with anti-SMAD7 (S7) antibody and immunoblotted with anti-HA-Ub antibodies. e [³⁵S]-methionine labeling and pulse-chase studies of SMAD7 in control (Co.sh) and OTUD1-depleted PC3 (#1 and #2). The amount of immunoprecipitated labeled protein after the chase was expressed as the percentage of that at the beginning of the chase (time 0) and shown in the right panel. Results are shown as means ± SD of two independent sets of experiments in duplicate. f IB of lysates derived from PC3 cells stably expressing empty vector (Co.vec), Myc-OTUD1-wt or OTUD1-CA and treated with cycloheximide (CHX, 20 μg/ml) at the indicated time points. Actin was analyzed as an internal loading control. Quantification of the band intensities was shown in the right panel. Band intensity was normalized to the t = 0 controls. Results are shown as means ± SD of three independent sets of experiments. *p < 0.05 (two-tailed Student′s t-test e, f)